Project 2 (Koch) SUMMARY/ABSTRACT Cardiac regeneration offers great promise for repair of injured myocardium and heart failure (HF), however it is not yet reality. This is due to cells not truly regenerating the heart as replacement myocytes. However, some benefit appears to occur in the post-ischemic heart with different stem cell injections, and data supports paracrine effects. Recent advances have suggested that the paracrine effects for any improvement in heart function after stem cell delivery is due to secreted exosomes from these cells. Exosomes are small vesicles containing specific cargo and it appears that they can carry potentially therapeutic molecules including growth factors and microRNAs (miRs). Data from our lab over the last two decades has implicated G protein-coupled receptor (GPCR) kinases (GRKs), especially GRK2, as key molecules in HF development following cardiac injury. Further, GRK2, which is up-regulated in failing human hearts, has emerged as a potential novel therapeutic target in HF. GRK2 has important effects on cardiac ?-adrenergic receptor (?AR) signaling that has crucial implications in HF. Importantly, inhibition of GRK2 with a peptide inhibitor (?ARKct) or knock-down of its expression leads to significant prevention and rescue of animal models of HF and preliminary data presented in this proposal shows that mechanisms of this repair includes increased markers of myocyte regeneration. Thus, we are interested in whether GRK2 or ?AR manipulation in myocytes or cardiac precursor cells (CPCs) may affect reparative or regenerative properties of the injured heart including examining paracrine, autocrine or exosome-based properties. We have started to investigate whether manipulation of GRK2 activity might alter exosomes and their cargo from either myocytes or CPCs, and whether exosomes from CPCs have improved regenerative properties. Indeed, we have recently found that introduction of the ?ARKct into CPCs improves survival and metabolism of these cells. ?ARKct-containing CPCs, as mediators of cardiac regeneration, will be employed to investigate whether manipulation of ?AR signaling and GRK2 activity may influence exosome-mediated cardiac repair in vivo. We will use mouse models and also move to the pig in order to carry out pre-clinical studies with the goal of future clinical translation. Specifically, the Central Hypothesis of this proposal is that inhibition or lowering of GRK2 in cardiac stem/precursor cells and/or cardiac myocytes improves myocardial repair through mechanisms that include favorable alterations of the content of secreted exosomes. This may include direct enhancement of ?-adrenergic signaling that improves survival and proliferation of these cells to positively affect cardiac regeneration. Specific Aims are: [1] To characterize the content of exosomes secreted from CPCs and myocytes with altered GRK2 expression and activity that can lead to changes in ?AR signaling, and to determine if altered exosome properties/cargo are responsible for the improved survival, metabolism and regenerative capacity of these cells; [2] To determine whether inhibiting GRK2 in CPCs improves exosome-based in vivo myocardial repair/regeneration and whether specific miR cargo in these GRK2-altered exosomes are involved in any therapeutic process; [3] To determine whether CPC exosomes from cells with lower GRK2 activity and ?ARKct-specific miR cargo enhance myocardial repair in a large animal, pre-clinical model of ischemic injury.